1. Field of the Invention
The present invention relates to G protein coupled receptors. More specifically, screening assays for Gs and Gq protein coupled receptor agonists and antagonists are provided. Also provided are stably transfected cell lines.
2. Related Art
Parathyroid hormone (PTH) is a major systemic regulator of bone turnover, and the closely related peptide, PTHrP, which is widely expressed in adult and fetal tissues, is believed to exert important paracrine actions, especially in developing cartilage and bone (Dempster, D. W., et al., Endocrine Rev 14:690-709 (1993); Lanske, B., et al., Science 273:663-666 (1996); Lee, K., et al., Endocrinology 137:5109-5118 (1996); Rixon, R. H., et al., J Bone Miner Res 9:1179-1189 (1994)). Exogenously administered PTH exerts striking effects upon bone mass in vivo, the nature of which depends critically upon the dose of PTH and the resulting temporal profile of the concentration of circulating peptide (Dempster, D. W., et al., Endocrine Rev 14:690-709 (1993)). Thus, continuous exposure to high PTH concentrations leads to net bone resorption and osteopenia, whereas intermittent administration of low doses leads to increased net bone formation--a finding that has fueled great interest in the possible use of PTH, or PTH analogs, as anabolic agents to increase bone mass and to prevent or treat metabolic bone diseases, including osteoporosis (Dempster, D. W., et al., Endocrine Rev 14:690-709 (1993), Whitfield, J. F., and Morley, P., Trends Pharmacol Sci 16:382-386 (1995)).
Both PTH and PTHrP can activate a single receptor, the PTH/PTHrP receptor (PTHR), which has been cloned from several species, including rat, opossum, mouse, pig and human, and shown to be expressed in cells of bone (Abou-Samra, A. B., et al., Proc Natl Acad Sci USA 89:2732-2736 (1992); Juppner, H., et al., Science 254:1024-1026 (1991); Schneider, H., Eur J Pharmacol 246:149-155 (1993); Bringhurst, F. R., et al., Endocrinology 132:2090-2098 (1991); Pines, M., et al., Endocrinology 135:1713-1716 (1994)). Activation of the PTHR in osteoblasts evokes multiple parallel signaling events, including activation of adenylyl cyclase (AC), phospholipase C (PLC) and cytosolic free calcium transients (Abou-Samra, A. B., et al., Proc Natl Acad Sci USA 89:2732-2736 (1992), Juppner, H., et al., Science 254:1024-1026 (1991), Bringhurst, F. R., et al., Endocrinology 132:2090-2098 (1993), Dunlay, R., and Hruska, K., Am J Physiol 258:F223-231 (1990); Fujimori, A., et al., Endocrinology 128:3032-3039 (1991); Yamaguchi, D. T., et al., J Biol Chem 262:7711-7718 (1987)). The links between each of these individual signaling events and the ultimate integrated tissue responses to PTH, such as changes in overall bone mass, remain largely undefined. It has been reported that PTH analogs which appear to selectively activate AC can generate a full anabolic effect on bone following intermittent administration in vivo (Rixon, R. H., et al., J Bone Miner Res 9:1179-1189 (1994), Whitfield, J. F., and Morley, P., Trends Pharmacol Sci 16:382-386 (1995), Whitfield, J. F., et al., Calcif Tissue Int 58:81-87 (1996)). Such observations have suggested that individual PTH second messengers may indeed be linked to specific tissue responses and, therefore, that the pattern of PTHR signaling events, as well as their intensity, may dictate both the qualitative and quantitative aspects of the response in bone. The issue is complicated by the fact that, in bone, mature osteoclasts are believed not to express PTHRs and thus must experience these influences of PTH only indirectly via responses generated by adjacent cells, such as osteoblasts or marrow stromal cells, which do express these receptors (Dempster, D. W., et al., Endocrine Rev 14:690-709 (1993), McSheehy, P., and Chambers, T., Endocrinology 118:824-828 (1986)). The manner whereby such osteoblastic or stromal PTH target cells might convey, to neighboring cells of the osteoclastic lineage, complex information reflecting subtle differences in temporal and concentration profiles of PTH exposure remains obscure.
Striking desensitization and downregulation of PTHRs has been described following exposure to high concentrations of ligand (Fujimori, A., et al., Endocrinology 128:3032-3039 (1991), Abou-Samra, A-B, et al., Endocrinology 129:2547-2554 (1991); Mitchell, J., and Goltzman, D., Endocrinology 126:2650-2660 (1990); Fukayama, S., et al., Endocrinology 131:1757-1769 (1992)) and it was reported recently that the pattern of signaling events generated by the rat PTHR is strongly influenced by the level of its expression on the cell surface (Guo, J., et al., Endocrinology 136:3884-3891 (1995)). Specifically, it was found that the magnitude of the PLC response was directly related to the density of available PTHRs on the surface of stably transfected LLC-PK1 cells across a range of expression (40,000-300,000 receptors per cell) that did not affect the maximal AC response (Guo, J., et al., Endocrinology 136:3884-3891 (1995)).
The human PTHR has been expressed previously in cultured cells (Pines, M., et al., Endocrinology 135:1713-1716 (1994), Schneider, H., et al., FEBS Lett 351:281-285 (1994), Pines, M., et al., Bone 18:381-389 (1996)), but its signaling properties have not yet been elucidated fully. In particular, the effects of alterations in human PTHR expression on the character of the signal transduction response(s) have not been systematically analyzed. Certain amino- or carboxyl-terminally truncated PTH analogs, such as PTH(3-34), PTH(7-34) and PTH(1-31), have been previously found to exhibit selective activation of only a subset of the usual PTHR second messengers (Rixon, R. H., et al., J Bone Miner Res 9:1179-1189 (1994), Whitfield, J. F., and Morley, P., Trends Pharmacol Sci 16:382-386 (1995), Fujimori, A., et al., Endocrinology 128:3032-3039 (1991), Abou-Samra, A. B., et al., Endocrinology 135:2588-2594 (1994); Azarani, A., et al., J Biol Chem 271:14931-14936 (1996); Chakravarthy, B. R., et al., Biochem Beefiest Res Commun 171:1105-1110 (1990); Fujimori, A., et al., Endocrinology 130:29-36 (1992); Jouishomme, H., et al., Endocrinology 130:53-60 (1992); Janulis, M., et al., Endocrinology 133:713-719 (1993)). The hPTH(1-31) analog was reported to activate AC but not PKC and yet to retain striking anabolic effects in ovariectomized rats (Rixon, R. H., et al., J Bone Miner Res 9:1179-1189 (1994), Jouishomme, H., et al., J Bone Miner Res 9:943-949 (1994)). Confirmation of such selective signaling via human PTHRs would provide important additional rationale for the development of these signal-specific PTH peptides for clinical use. Thus, there is a need in the art for characterization of responses mediated by PTHR.